Human papillomavirus (HPV) is a class of small non-enveloped DNA virus; currently more than 100 genotypes of HPVs have been identified. HPV primarily infects human host through squamous and mucosal epithelia to cause benign and malignant epithelial neoplasia. Based on tissue-tropism of HPVs, they can be classified into cutaneous type and mucosal type. The mucosal type of HPVs can be further divided into two subtypes according to their relationships with benign and malignant epithelial neoplasia: the low-risk type, which is associated with development of precondyloma acuminate and other benign lesions, and the high-risk type, which can give rise to precancerous lesions of cervical cancer and other cancers. Studies have shown that persistent infection of the high-risk type HPV is causally linked to cervical cancer and HPV positives can be detected in about over 99.7% cervical cancer patients. [Walboomers, J. M. et al. J. Pathol. 1999, 189:12-19]. In addition, above 90% of anus and vaginal squamous carcinoma, 40% of vulva and penile cancer, 12% of oropharyngeal cancer and 3% of oral cancer are associated with infections of the high-risk type HPVs [Lacey C J N. Medicine, 2005, 33(10):51-55]. The International Agency for Research on Cancer (IARC) analyzed 3607 cervical cancer samples obtained from 25 countries of the world (excluding East Asia) and 15 high-risk types of HPV were found. Based on the positive infection rate, among the highest were HPV 16(53.5%), 18(17.2%), 45(6.7%), 31(2.9%), 33(2.6%), 52(2.3%), and 58(2.2%), respectively [Munoz, N. et al. Int. J. Cancer, 2004, 111: 78-285]. Research from our team and others in China showed that HPV 16 was the most frequently detected type among Chinese patients with pre-cancerous lesions and cervical cancer, while HPV 58 and HPV 18 were next to HPV 16. For example, Chen et al. found that in Sichuan province, the infection frequencies of HPV 16, 58 and 18 in patients with cervical cancer were 78.6%, 20.0% and 9.7%, respectively; studies by Liu et al. with 815 cervical cancer samples from fourteen provinces in China also demonstrated that HPV 16 positive was most frequently detected and HPV 58 positive ranked the second; in addition, the positive frequencies of HPV 58 in cervical cancer patients was 12.3-17.5% in Northeastern China, Southeastern China and Shanxi province, 12.3-19.9% in Taiwan and 31.5-33.3% in Hong Kong [Si J Y, Lee K, Han R C, et al. J Cancer Res Clin Oncology. 1991, 117: 454-459; Liu B Y, Li J, De Villiers E M et al. Chinese Journal of Experimental and Clinical Virology, 1996, 10:118-121; Hao L, Ma Y Y, Mo J S, et al. Gynecologic Oncology, 2006, 101(1):40-45; Chan P K, Li W H, Chan M Y, et al. J Med Virol. 1999, 59 (2):232-238; Chen C A, Liu C Y, Chou H H, Chou C Y, et al. Int J Gynecol Cancer. 2006, 16(5):1801-8]. The above studies demonstrated that besides HPV 16, HPV 58 is another important etiologic cause for cervical cancer among Chinese women, i.e. HPV 16, 58 and 18 are among the viral genotypes that are closely associated with the development of cervical cancer in China. Infection of HPV 58 is very common in Japan, Korea and other East Asian countries. Currently, 12 low-risk type HPVs (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 and CP6108) have been identified; infections of these HPVs generally cause genital warts. Condyloma acuminate is a common type of genital warts and is the second highest-occurring sexually transmitted disease in China (next to gonorrhea). It is mainly transmitted via sexual routes. Over the past decade, the morbidity of condyloma acuminate increased constantly. Research has demonstrated that in tissues of Chinese patients with condyloma acuminate, 82% were HPV 6 positive and 6% were HPV 11 positive.
Currently there is limitation in clinical therapeutics for condyloma acuminate and later stage cervical cancer. There is no effective vaccine in the Chinese domestic market. Pap smear screen for cervical cancer is relatively expensive and is only suitable for economically advanced area. Although effective methods for detecting HPV infections are available and the majority cervical cancer patients can be treated, among the detected HPV positives, about 35% patients would still eventually develop persistent infections or tumors. In rural developing area where cervical cancer possesses high incidence rates, pap smear screen cannot be widely applied due to economic and cultural reasons. Therefore, develop effective prophylactic vaccination provides a better solution over pap smear screen in preventing cervical cancer.
L1 protein may be self-assembled into virus-like particles (VLPs) when expressed in vitro. Similar to naturally-occurring viruses, VLPs may induce protective neutralizing antibodies. However, such antibodies are highly genotype-specific and usually have no cross-neutralizing activities. Thus, vaccination with one HPV VLP genotype is not able to prevent the infection of other HPV genotypes. Clinical studies showed that both vaccines have high safety and immune protection effects [Harper D M, et al. Lancet. 2004, 364:1757-1765; Harper D M et al. The Lancet. 2006; Internet: 1-9]. However, since none of the two vaccines contains HPV58, they do not meet the requirements for preventing HPV-infection associated diseases in China and other East Asian countries. Chinese patent application CN 1869215A has released modified HPV16, 58 and 6 L1 genes. In addition, given that HPV-infection and infection-associated diseases have high incidence rates in developing countries, the key point of successfully developing HPV vaccines would be producing high expression level of the L1 protein in vitro, thus reduce the cost of vaccine manufacturing.
However, wild type HPV L1 (L1wt) genes amplified from patients possess several characters including preferred codon usage bias, hypothetical transcription stop codons (e.g. AT rich structures such as TATATA, TAGATA, and TACATA), long GC rich stems of mRNAs and their complex secondary structures. Examples including the existence of RNA transcription inhibiting element that located at the N-terminus of wild type HPV 16 L1. These gene characters result in low expression level of the L1 protein in vitro, prohibit large-scale VLP production and further prohibit the manufacturing of low-cost vaccines. To solve the limitation of the current technology, the present invention provides optimized HPV 16, 58, 18, 6 and 11 L1 genes that may produce high level gene and protein expressions of L1 in insect cells, and obtain HPV 16, 58, 18, 6 and 11 VLPs. The present invention also provides multivalent vaccines comprising the above said five genotypes of HPV VLPs suitable for prevention and treatment of HPV-infection associated diseases in China and other East Asian countries.